In the somatic cells of the normal human female, one of the two X chromosomes is genetically inactivated. Several mechanisms have been proposed to explain X-inactivation, however, there is no experimental evidence in support of any of these hypotheses. One proposed mechansims involves DNA modification by methylation. By specifically altering the methylation pattern of DNA and determining the resulting changes on the expression of genes located on the inactive human X chromosome, we propose to test the hypothesis that X-inactivation may be mediated by methylation of DNA. The only methylated base found in mammalian DNA is 5-methylcytosine. It has recently been shown that incorporation of 5-azacytidine (a cytidine analog) into DNA, leads to hypomethylation of DNA. This is thought to result from lack of methylation at the sites of incorporation of 5-azacytidine, which has a substitution of the C at position 5 in the pyrimidine by N. It has also been shown that treatment of mouse fibroblast cultures with 5-azacytidine leads to expression of differentiated functions, otherwise not expressed. From the fusion of HPRT (hypoxanthine phosphoribosyltransferase) deficient mouse cells and human cells containing a balanced X-autosome translocation, we have isolated a hybrid clone containing a structurally normal inactive human X chromosome (but not an active X). This hybrid clone does not express human PGK or G6PD (known human X-markers). We propose to treat this clone with 5-azacytidine and evaluate the expression of human X-linked markers in these mouse-human hybrid cells. Reactivation of human HPRT locus can be monitored by selection of these cells in HAT medium, following treatment. Expression of human X markers following 5-azacytidine treatment will support the hypothesis that X-anactivation may be mediated by DNA methylation.